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[Entomopoxviruses and biological control.].

Poxviridae are divided into two subfamilies: the Chordopoxvirinae (poxviruses of vertebrates) and the Entomopoxvirinae (insect poxviruses). Entomopoxviruses (EPVs) are large (300-400 nm) oval shaped viruses. The genome of EPVs is large, with covalent ends and is a linear double-stranded DNA (200-240 kbp) molecule. The Entomopoxvirinae comprises three genera based on host insect and virion morphology. Genus A viruses infect coleopterans, genus B viruses infect lepidopterans and orthopterans, and genus C viruses infect dipterans. The Melolontha melolontha entomopoxvirus (MmEPV) was the first poxvirus to be described as being associated with an insect disease. Then, several entomopoxviruses (EPVs) have been found to infect Coleoptera, Lepidoptera, Diptera, Hymenoptera, and Orthoptera. Entomopoxviruses are very similar to orthopoxvirus and molluscipoxvirus that cause dermal lesions and pox diseases in humans. Therefore, these viruses have great importance in understanding their replication mechanism as well as in the use as a gene expression vector and as a pest control agent. In this review article, we present information about entomopoxviruses on which important studies have been done recently.

English Abstract↗

A putative DNA helicase and novel oligoribonuclease in the Diachasmimorpha longicaudata entomopoxvirus (DlEPV).

Diachasmimorpha longicaudata entomopoxvirus (DlEPV) is a symbiotic entomopoxvirus (EPV) of the parasitic wasp Diachasmimorpha longicaudata. It has a double-stranded DNA genome of 250-300 kb and is >60% A-T rich. We describe ten ORFs (RI-35-1 to -10) contained within a 5.64 kb clone, RI-35, from a DlEPV EcoRI genomic library. Our goal was to identify unique motifs and compare them with others in the database, particularly those of poxviruses. Two ORFs (RI-35-1 and RI-35-7, respectively) encode putative proteins (113 aa and 219 aa) that are probably involved in regulating gene expression based on their predicted nuclear localization and the presence of SPxx motifs, leucine-zipper like sequences (113 aa), and a basic domain (219 aa). The largest gene (RI-35-3) is under the control of an intermediate/late promoter and is presumed to encode a cytoplasmic 480 aa DNA-dependent DNA helicase with conserved motifs that are characteristic of DExH helicases. Amino acid analysis of the DNA helicase sequence showed that DlEPV is close to but distinct from the Genus B EPVs. The DlEPV helicase is also distinct from that of the Diadromus pulchellus ascovirus 1a from the D. pulchellus parasitic wasp, with less than 10% amino acid identity. DlEPV encodes a 207 aa oligoribonuclease (RI-35-8) of the DEDDh family of exoribonucleases. The second largest ORF (RI-35-9) is under the control of a poxvirus early promoter and encodes a protein of 329 aa that is likely DlEPV-specific. Three ORFs (RI-35-4, -5, and -6) overlap (in the anti-sense strand) with ORFs encoding putatively important virus replication proteins (which were also under the control of intermediate promoters) and are presumably not expressed in DlEPV. These results support earlier reports that DlEPV is a member of the sub-family Entomopoxvirinae, most likely in Group C, and is the first symbiotic EPV described to date from a parasitic wasp.

Amino Acid Sequence↗

The spheroidin of an entomopoxvirus isolated from the grasshopper Anacridium aegyptium (AaEPV) shares low homology with spheroidins from lepidopteran or coleopteran EPVs.

Based on virion morphology, the current virus taxonomy groups entomopoxviruses (EPVs) (Poxvirus: Entomopoxvirinae) from coleopteran and dipteran hosts in separated genera, wilts it keeps viruses infecting either lepidopteran or orthopteran hosts in the same genus. In contrast to the morphological criteria, the few data available from recent studies at the genetic level have suggested that EPVs infecting different insect orders are phylogenetically distant. In order to elucidate EPVs phylogeny we have cloned and sequence the highly conserved/highly expressed spheroidin gene of Anacridium aegyptium entomopoxvirus (AaEPV). This gene and its promoter is of interest for the development of genetic engineering on EPVs. The spheroidin gene was located in the AaEPV genome by Southern blot and hybridisation with specific degenerated oligonucleotides probes synthesised after partial sequencing of the purified spheroidin protein. A total of 3489 bp were sequenced. This sequence included the coding and promoter region of 969 residues 108. 8 kDa protein identified as spheroidin. AaEPV spheroidin contains 21 cysteine residues (2.2%) and 14 N-glycosylation putative sites distributed along the sequence. The cysteine residues are particularly abundant at the C-terminal end of the protein, with 11 residues in the last 118 aa. Our results confirm that the spheroidin is highly conserved only between EPVs isolated from the same insect order. Polyclonal antibodies raised against AaEPV spherules specifically revealed spheroidin in Western Blots failing to cross-react with MmEPV or AmEPV spheroidins or MmEPV fusolin. Comparison of spheroidins at the aa level demonstrate that AaEPV spheroidin shares only 22.2 and 21.9% identity with the lepidopteran AmEPV and the coleopteran MmEPV spheroidins, respectively, but 82.8% identity with the orthopteran MsEPV spheroidin. Only two highly conserved domains containing the sequence (V/Y)NADTG(C/L) and LFAR(I/A) have been identified in all known spheroidins. The phylogenetic tree constructed according to the CLUSTALX analysis program revealed that EPVs are clearly separated in three groups - lepidopteran, coleopteran and orthopteran - according to the insect order of the virus hosts. In base to our results, the split of the genus Entomopoxvirus B dissociating lepidopteran and orthopteran EPVs into two different genera is suggested.

Amino Acid Sequence↗

Virions of Heliothis armigera entomopoxvirus contain a homologue of the vaccinia VP8 major core protein.

An antigenic 30 K virion protein of Heliothis armigera entomopoxvirus (HaEPV) has been identified as a homologue of the chordopoxvirus (ChPV) VP8 major virion core protein. Like its homologue in vaccinia virus, the mature HaEPV 30 K protein is derived by post-translational cleavage of a precursor at a conserved AGA motif. The HaEPV 30 K protein is the first EPV structural virion protein to be described, and elucidation of its characteristics provides evidence for the assumption that morphological similarities observed between virions of the sub-families Entomopoxvirinae and Chordopoxvirinae by microscopy reflect corresponding similarities at a molecular level. Sequencing of the HaEPV genome adjacent to the 30K locus identified an ORF encoding a homologue of the regulatory sub-unit of the ChPV poly(A) polymerase enzyme; the conceptual product of this ORF showed 25-31% aa sequence identity to those of various ChPVs. The presence of this gene in the HaEPV genome supports the hypothesis that there is a substantial correspondence in basic metabolic processes of members of the two poxvirus sub-families, despite their utilization of divergent host groups. In contrast, the relative positions of the 30 K and poly(A) polymerase loci in the HaEPV genome provide further evidence of substantial genomic re-arrangement subsequent to divergence of these viral taxa.

Amino Acid Sequence↗

The genome of Melanoplus sanguinipes entomopoxvirus.

The family Poxviridae contains two subfamilies: the Entomopoxvirinae (poxviruses of insects) and the Chordopoxvirinae (poxviruses of vertebrates). Here we present the first characterization of the genome of an entomopoxvirus (EPV) which infects the North American migratory grasshopper Melanoplus sanguinipes and other important orthopteran pests. The 236-kbp M. sanguinipes EPV (MsEPV) genome consists of a central coding region bounded by 7-kbp inverted terminal repeats and contains 267 open reading frames (ORFs), of which 107 exhibit similarity to previously described genes. The presence of genes not previously described in poxviruses, and in some cases in any other known virus, suggests significant viral adaptation to the arthropod host and the external environment. Genes predicting interactions with host cellular mechanisms include homologues of the inhibitor of apoptosis protein, stress response protein phosphatase 2C, extracellular matrixin metalloproteases, ubiquitin, calcium binding EF-hand protein, glycosyltransferase, and a triacylglyceride lipase. MsEPV genes with putative functions in prevention and repair of DNA damage include a complete base excision repair pathway (uracil DNA glycosylase, AP endonuclease, DNA polymerase beta, and an NAD+-dependent DNA ligase), a photoreactivation repair pathway (cyclobutane pyrimidine dimer photolyase), a LINE-type reverse transcriptase, and a mutT homologue. The presence of these specific repair pathways may represent viral adaptation for repair of environmentally induced DNA damage. The absence of previously described poxvirus enzymes involved in nucleotide metabolism and the presence of a novel thymidylate synthase homologue suggest that MsEPV is heavily reliant on host cell nucleotide pools and the de novo nucleotide biosynthesis pathway. MsEPV and lepidopteran genus B EPVs lack genome colinearity and exhibit a low level of amino acid identity among homologous genes (20 to 59%), perhaps reflecting a significant evolutionary distance between lepidopteran and orthopteran viruses. Divergence between MsEPV and the Chordopoxvirinae is indicated by the presence of only 49 identifiable chordopoxvirus homologues, low-level amino acid identity among these genes (20 to 48%), and the presence in MsEPV of 43 novel ORFs in five gene families. Genes common to both poxvirus subfamilies, which include those encoding enzymes involved in RNA transcription and modification, DNA replication, protein processing, virion assembly, and virion structural proteins, define the genetic core of the Poxviridae.

Amino Acid Sequence↗

Amsacta moorei Entomopoxvirus inhibitor of apoptosis suppresses cell death by binding Grim and Hid.

Inhibitor of apoptosis (iap) genes have been identified in the genomes of two independent families of insect viruses, the Baculoviridae and the Entomopoxvirinae. In this report, we examined the functional attributes of the Amsacta moorei entomopoxvirus-encoded IAP protein (AMV-IAP). The binding specificity of the individual baculoviral IAP repeat (BIR) domains of AMV-IAP was investigated by using a random-peptide, phage display library, and sequences similar to the amino termini of proapoptotic Drosophila proteins in the Reaper/Hid/Grim family were identified. Furthermore, the BIR domains of AMV-IAP protein were demonstrated to bind the mammalian IAP inhibitor Smac through the AVPI tetrapeptide sequence, suggesting that the peptide binding pocket and groove found in the insect and mammalian IAPs is conserved in this viral protein. Interaction analysis implicated BIR1 as the high-affinity site for Grim, while BIR2 interacted more strongly with Hid. Both Grim and Hid were demonstrated to interact with AMV-IAP in vivo, and Grim- or Hid-induced cell death was suppressed when AMV-IAP was coexpressed.

Animals↗

The non-permissive infection of insect (gypsy moth) LD-652 cells by Vaccinia virus.

The members of Poxviridae family are among the most complex of animal viruses and subfamily members infect both vertebrate (Chordopoxvirinae) and invertebrate (Entomopoxvirinae) hosts, respectively. Vaccinia virus (VV) is the most commonly studied vertebrate virus and the entomopoxvirus of Amsacta moorei (AmEPV) is the prototypic insect virus. AmEPV, while not able to productively infect vertebrate cells, does enter vertebrate cells and expresses early genes after which the infection aborts although the cells survive (Y. Li, R. L. Hall, and R. W. Moyer. J.Virol. 71(12), 95579562, 1997). We show here that a recombinant VV, containing the lacZ gene regulated by the cowpox virus A-type inclusion (ATI) late promoter, likewise does not productively infect insect cells. Our results suggest that the recombinant VV enters insect cells, host protein synthesis is inhibited, early gene expression is normal, and viral DNA replication occurs as does late protein synthesis. However, little if any proteolytic processing of late viral proteins, typical of morphogenesis, is observed. Electron micrographs of infected cells suggest that while cytoplasmic virosomes (factories) are formed, there is little indication of further morphogenesis or any formation of mature virions. Therefore, while both orthopoxviruses and entomopoxviruses fail to replicate in heterologous hosts, the nature of abortive infections is quite different.

Animals↗

Genome-based phylogeny of poxvirus.

A comprehensive phylogenetic analysis of Poxviridae was performed in this study. Phylogenetic trees were reconstructed from whole genomic information including gene order, gene content, and all conserved gene protein sequences. Gene content and gene order were the methods used for the first time in cladogram reconstruction of poxvirus. The results of the different methods finally shared mainly consistent tree topologies, which were supported by the classic taxonomy of Poxviridae. According to these topologies, the Entomopoxvirinae and Chordopoxvirinae subfamilies are divergent from each other. Subfamily Chordopoxvirinae could be divided into four main groupings, which could be further divided into subgroups. Parapoxvirus and Molluscipoxvirus have been considered distinct from other Chordopoxvirus. Results from our study consistently supported this suggestion. In our results, Parapoxvirus and Molluscipoxvirus were the most closely related genera and formed a group. This group could be the one that branched after the divergence of Avipoxviruses. Additionally, rabbitpox virus and vaccinia virus steadily formed clusters with high bootstrap support in all the cladograms.

Base Sequence↗

Detection of a virus enhancing factor in the spheroid, spindle, and virion of an entomopoxvirus.

Spheroids, spindles, and virions of an entomopoxvirus (EPV) enhanced the infectivity of a nuclear polyhedrosis virus (NPV) when they were perorally administered to larvae of the armyworm, Pseudaletia separata. Spheroids and spindles at the same dose exhibited nearly the same enhancing activity. When the dose of spheroids or spindles was reduced 10 times, the median infectious dose of the NPV was increased approximately 100 times. An antiserum against an enhancing factor detected the homologous antigen in spheroids, spindles, and tissue-derived EPV virions but not in spheroid-derived virions.

Animals↗

A new entomopoxvirus from a cockroach: light and electron microscopy.

The cockroach entomopoxvirus caused a chronic infection in cultures of the German cockroach Blattella germanica. Heavily infected specimens showed a reduced mobility. Ellipsoid virus occlusion bodies (8 x 5 to 19 x 12 microm) were found intracellularly in tracheole cells, in the hypodermis, in fat body cells, and in muscles. Several hundred virus particles were integrated in a single occlusion body (OB), their long axis being oriented axially. Ovoid viroids measured 320 x 190 nm and possessed a unilateral, concave core and one lateral body. Starting occlusion, small granules attached to the virus particles which later transformed to a beaded, wavy envelope. An initial halo around the occluded virions disappeared in more central regions of the OB. Virus particles were formed either in a dense cytoplasmic area containing electron-dense viroids, or in a loosely aggregated viroplasm. In the latter, developmental stages were mainly represented by spheres with double membranes enclosing granular material. Spindles and larger crystal-like virus-free inclusion bodies occurred in the cytoplasm. The cytoplasm of infected cells appeared degenerated and the chromatin of the nuclei condensed at the periphery or disintegrated. Taxonomically, the described virus exhibits features of both EPV genus A and EPV genus B. Provisory it is named Blattella germanica EPV (BgEPV). A possible use of the cockroach EPV as a biological control agent is discussed.

Animals↗

Spindle bodies of Heliothis armigera entomopoxvirus develop in structures associated with host cell endoplasmic reticulum.

Immunoelectron microscopy has shown that morphogenesis of spindle bodies (SB) of Heliothis armigera entomopoxvirus involves an iterative process of condensation, aggregation, and crystallization of the major constituent protein (fusolin) within the perinuclear space and endoplasmic reticulum (ER) of infected cells and in vesicles derived from ER constituents. The ER-specific chaperone BiP has been observed to be associated with developing SBs at all stages of this process, and it is postulated that its sequestration within these bodies may have consequences for host cell metabolism.

Animals↗

Enhanced fusion of a nucleopolyhedrovirus with cultured cells by a virus enhancing factor from an entomopoxvirus.

Fusion of Pseudaletia unipuncta nucleopolyhedrovirus with an armyworm cell line (SIE-MSH-805-F) was studied by means of three fluorescence assays that are based on the relief of fluorescence self-quenching of octadecylrhodamine B chloride (R18). A gradual increase in fluorescence intensity indicative of virus-cell fusion was observed by spectrofluorometry when R18-labeled polyhedron-derived virus was incubated with cultured cells. The fusion was enhanced by the virus enhancing factor (EF) from Pseudaletia separata entomopoxvirus. Lysosomotropic agents had little effect on the virus-cell fusion. The percentage of positively fluorescent cells, as determined by flow cytometry, gradually increased after the addition of labeled virus and was higher in the presence of the EF than in its absence. Confocal microscopy of cultured cells that had been combined with labeled virus showed that the fluorescence appeared first on their surface. The plasma membrane of cultured cells had specific affinity to the EF, as revealed by indirect immunofluorescence microscopy.

Animals↗

A bacterially produced virus enhancing factor from an entomopoxvirus enhances nucleopolyhedrovirus infection in armyworm larvae.

Using an Escherichia coli expression system, pGEX-2T, that expresses foreign sequences as fusion proteins with a glutathione S-transferase (GST) carrier, we have expressed a virus enhancing factor (EF) from Pseudaletia separata entomopoxvirus, which enhances P. unipuncta multi nucleopolyhedrovirus (PsunMNPV) infection in larvae of the armyworm, P. separata. The lysates of transformed E. coli cells, which were not active in enhancing PsunMNPV infection, became active when treated with either trypsin or thrombin. The GST-EF fusion protein in a lysate was purified with a bulk GST purification module and cleaved into the EF and GST moieties with thrombin. Removal of the GST moiety with glutathione-Sepharose 4B resulted in a highly purified EF preparation, which enhanced PsunMNPV infection in armyworm larvae and PsunMNPV fusion with an armyworm cell line, SIE-MSH-805-F.

Animals↗

Cloning and sequencing of the spherulin gene, the occlusion body major polypeptide of the Melolontha melolontha entomopoxvirus (MmEPV).

In the late stage of infection, virions of the Melolontha melolontha entomopoxvirus (MmEPV) are occluded into cytoplasmic paracrysalline proteinaceous occlusion bodies designated spherules (A. Amargier, C. Vago, G. Meynadier, 1964, Mikroskopie 19, 309-315). We have cloned and sequenced a 4-kpb DNA fragment of the MmEPV genome encompassing the spherule major protein gene named spherulin. The spherulin gene contains an open reading frame able to code for a 942-amino-acid (aa) polypeptide (MW 109 kDa), consistent with a size above 100 kDa determined by SDS-PAGE for purified spherulin. The MmEPV spherulin showed more than 40% as homology with the Amsacta moorei EPV (AmEPV) spheroidin and shared homologies with the partially sequenced Choristoneura biennis EPV (CbEPV) spheroidin, indicating that this biologically important polypeptide is well conserved among EPVs infecting phylogenetically as distant groups of insects as lepidoptera and coleoptera. Western blot analyses confirmed the relationships between the three polypeptides. In contrast, no homology was detected between the MmEPV spherulin and EPV fusolins or vertebrate poxvirus A-type inclusion proteins. The 45 bases upstream from the ATG initiation codon of spherulin shared 60% homology with the vaccinia virus late promoters including the highly conserved TAAATG consensus sequence. Furthermore, the 5' extremity of the spherulin mRNA consisted of a poly(A) tract of about 20 nucleotides just upstream from the AUG translational initiation codon. These are characteristic features of vertebrate poxvirus late mRNAs suggesting similar modalities of gene expression for vertebrate and insect poxvirus genomes.

Amino Acid Sequence↗

The Melolontha melolontha entomopoxvirus (MmEPV) fusolin is related to the fusolins of lepidopteran EPVs and to the 37K baculovirus glycoprotein.

We have cloned and sequenced a 1.7-kbp DNA fragment of the MmEPV genome encompassing the major polypeptide of the spindle-shaped inclusions gene termed fusolin. The sequence contained a single open reading frame of 1203 nt capable of coding for a polypeptide of 45.8 kDa. The 13 N-terminal amino acid (aa) residues were hydrophobic and could act as a signal peptide. The aa sequence also contained 13 cysteine residues very likely involved in paracrystal formation. This sequence showed significant homologies with the fusolins of two lepidopteran EPVs, the Choristoneura biennis EPV (CbEPV) and the Heliothis armigera EPV, and also with the 37K glycoproteins of Autographa californica and Orgyia pseudotsugata baculoviruses. No homology was found between the MmEPV fusolin and the 100K MmEPV spherulin, nor with the 110K polypeptide of the CbEPV and Amsacta moorei EPV spheroidins. These data were confirmed by Western blot analysis. Transfection of vaccinia-infected mammalian cells with a plasmid encompassing the fusolin sequence plus the upstream regulatory region resulted in transient expression of the gene. This indicated that the vaccinia transcription machinery is able to transcribe the fusolin gene. The fusolin was also expressed in insect cells via a recombinant baculovirus.

Amino Acid Sequence↗

The effect of inhibitors on the growth of the entomopoxvirus from Amsacta moorei in Lymantria dispar (gypsy moth) cells.

Within the family of Poxviridae, the entomopoxviruses are the most distant relatives of the more well-known and intensively studied orthopoxviruses (vaccinia and variola). The growth of the entomopoxvirus from Amsacta moorei (AmEPV) has been characterized in cell culture and compared to that of vaccinia virus (VV), the prototypic orthopoxvirus. The overall characteristics of infected cell cultures were generally similar between the two viruses. One striking difference noted was the apparent absence of proteolytic processing of late AmEPV viral proteins, a hallmark of vertebrate poxvirus infections associated with viral morphogenesis. AmEPV, like VV, was found to be sensitive to all the inhibitors of viral infection tested including phosphonoacetic acid, 1-beta-D-arabinofuranosylcytosine, and isatin-beta-thiosemicarbazone (IBT), a compound associated with the rather specific inhibition of vertebrate poxviruses. While both VV and AmEPV are inhibited by IBT, the inhibition of AmEPV, unlike that of VV, is not accompanied by either a breakdown of ribosomal RNA or a global inhibition of late viral protein synthesis. Instead, in the presence of IBT, AmEPV enveloped, immature virions form devoid of a well-differentiated core, which unlike mature virions fail to insert into occlusion bodies.

Animals↗

The Amsacta moorei entomopoxvirus spheroidin gene is improperly transcribed in vertebrate poxviruses.

The Amsacta moorei entomopoxvirus (AmEPv) spheroidin is the most highly expressed late viral gene product in infected insect cells. However, when a cassette containing the spheroidin gene and putative promoter was inserted into cowpox (CPV) or vaccinia viruses, only very low levels of spheroldin gene expression were observed. Primer extension analysis suggests much lower spheroidin gene transcript levels than seen either for the highly expressed CPV A-type inclusion gene or for the spheroidin gene within infected insect cells, indicating that in vertebrate cells, the spheroidin promoter functions poorly if at all. Examination of the spheroidin mRNA synthesized in recombinant CPV shows that the 5' start site of the spheroidin transcript was also unexpectedly imprecise and upstream (approximately 31 bp) of the well-defined start site normally observed in AmEPV-infected insect cells. Sequencing of the 5' terminus of the CPV recombinant spheroidin mRNA suggested that 5' poly(A), a characteristic feature of late poxvirus mRNAs and spheroidin mRNA derived from insect cells, was absent, despite the presence of the typical vertebrate poxvirus late promoter consensus sequence, TAAATG. Our results indicate that insect and vertebrate poxvirus promoters may not be universally interchangeable and imply that there are regulatory features of gene expression unique to the infected insect cell environment.

Animals↗

Characterization of a DNA topoisomerase encoded by Amsacta moore entomopoxvirus.

We have identified an Amsacta moorei entomopoxvirus (AmEPV) gene encoding a DNA topoisomerase. The 333-amino acid AmEPV topoisomerase displays instructive sequence similarities to the previously identified topoisomerases encoded by five genera of vertebrate poxviruses. One hundred nine amino acids are identical or conserved among the six proteins. The gene encoding AmEPV topoisomerase was expressed in bacteria and the recombinant enzyme was partially purified. AmEPV topoisomerase is a monomeric enzyme that catalyzes the relaxation of supercoiled DNA. Like the vaccinia, Shope fibroma virus, and Orf virus enzymes, the AmEPV topoisomerase forms a covalent adduct with duplex DNA at the target sequence CCCTT decreases. The kinetic and equilibrium parameters of the DNA cleavage reaction of AmEPV topoisomerase (Kobs = 0.08 sec-1; Kcl = 0.22) are similar to those of the vaccinia virus enzyme.

Amino Acid Sequence↗